The stereochemistry of trans-4-hydroxynonenal-derived exocyclic 1,N2-2'-deoxyguanosine adducts modulates formation of interstrand cross-links in the 5'-CpG-3' sequence

The trans-4-hydroxynonenal (HNE)-derived exocyclic 1, N(2)-dG adduct with (6S,8R,11S) stereochemistry forms interstrand N(2)-dG-N(2)-dG cross-links in the 5'-CpG-3' DNA sequence context, but the corresponding adduct possessing (6R,8S,11R) stereochemistry does not. Both exist primarily as diastereomeric cyclic hemiacetals when placed into duplex DNA [Huang, H., Wang, H., Qi, N., Kozekova, A., Rizzo, C. J., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 10898-10906]. To explore the structural basis for this difference, the HNE-derived diastereomeric (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were examined with respect to conformation when incorporated into 5'-d(GCTAGC XAGTCC)-3' x 5'-d(GGACTCGCTAGC)-3', containing the 5'-CpX-3' sequence [X = (6S,8R,11S)- or (6R,8S,11R)-HNE-dG]. At neutral pH, both adducts exhibited minimal structural perturbations to the DNA duplex that were localized to the site of the adduction at X(7) x C(18) and its neighboring base pair, A(8) x T(17). Both the (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were located within the minor groove of the duplex. However, the respective orientations of the two cyclic hemiacetals within the minor groove were dependent upon (6S) versus (6R) stereochemistry. The (6S,8R,11S) cyclic hemiacetal was oriented in the 5'-direction, while the (6R,8S,11R) cyclic hemiacetal was oriented in the 3'-direction. These cyclic hemiacetals effectively mask the reactive aldehydes necessary for initiation of interstrand cross-link formation. From the refined structures of the two cyclic hemiacetals, the conformations of the corresponding diastereomeric aldehydes were predicted, using molecular mechanics calculations. Potential energy minimizations of the duplexes containing the two diastereomeric aldehydes predicted that the (6S,8R,11S) aldehyde was oriented in the 5'-direction while the (6R,8S,11R) aldehyde was oriented in the 3'-direction. These stereochemical differences in orientation suggest a kinetic basis that explains, in part, why the (6S,8R,11S) stereoisomer forms interchain cross-links in the 5'-CpG-3' sequence whereas the (6R,8S,11R) stereoisomer does not.     

Interstrand cross-linking reaction in triplexes containing a monofunctional transplatin-adduct.

Our aim was to determine whether a single transplatin monofunctional adduct, either trans-[Pt(NH3)2(dC)Cl]+ or trans-[Pt(NH3)2(dG)Cl]+ within a homopyrimidine oligonucleotide, could further react and form an interstrand cross-link once the platinated oligonucleotide was bound to the complementary duplex. The single monofunctional adduct was located at either the 5' end or in the middle of the platinated oligonucleotide. In all the triplexes, specific interstrand cross-links were formed between the platinated Hoogsteen strand and the complementary purine-rich strand. No interstrand cross-links were detected between the platinated oligonucleotides and non-complementary DNA. The yield and the rate of the cross-linking reaction depend upon the nature and location of the monofunctional adducts. Half-lives of the monofunctional adducts within the triplexes were in the range 2-6 h. The potential use of the platinated oligonucleotides to modulate gene expression is discussed.     

Intrastrand cross-links are not formed in the reaction between transplatin and native DNA: relation with the clinical inefficiency of transplatin.

The reaction between trans-diamminedichloroplatinum(II) and single-stranded oligonucleotides containing the sequence d(GXG) (X being an adenine, cytosine or thymine residue) yields trans-[Pt(NH3)2[(GXG)-GN7,GN7]] intrastrand cross-links. These cross-links do not prevent the pairing of the platinated oligonucleotides with their complementary strands but they decrease the thermal stability of the duplexes. The thermal stability is not much affected by the chemical nature of the X residue and its complementary base. By gel electrophoresis, it is shown that the trans- [Pt(NH3)2[d(GTG)-GN7,GN7]] cross-link bends the DNA double helix (26 degrees) and unwinds it (45 degrees). The pairing of the platinated oligonucleotides with their complementary strands promotes the rearrangement of the 1,3-intrastrand cross-links into interstrand cross-links. At a given temperature, the nature of the X residue, its complementary base and of the base pairs adjacent to the adducts do not dramatically affect the rate of the reaction. To know whether trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links do not rearrange in some sequences, the location of these adducts was searched in double-stranded DNA after reaction with trans-diamminedichloroplatinum(II) by means of the 3'-5' exonuclease activity of T4 DNA polymerase. At low level of platination, trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links were not detected. Monofunctional adducts and interstrand cross-links were mainly formed. These results are discussed in relation with the clinical inefficiency of trans-diamminedichloroplatinum(II).     

Conformational analysis of site-specific DNA cross-links of cisplatin-distamycin conjugates

The requirement for novel platinum antitumor drugs led to the concept of synthesis of novel platinum drugs based on targeting cisplatin to various carrier molecules. We have shown [Loskotova, H., and Brabec, V. (1999) Eur. J. Biochem. 266, 392-402] that attachment of DNA minor-groove-binder distamycin to cisplatin changes several features of DNA-binding mode of the parent platinum drug. Major differences comprise different conformational changes in DNA and a considerably higher interstrand cross-linking efficiency. The studies of the present work have been directed to the analysis of oligodeoxyribonucleotide duplexes containing single, site-specific adducts of platinum-distamycin conjugates. These uniquely modified duplexes were analyzed by Maxam-Gilbert footprinting, phase-sensitive gel electrophoresis bending assay and chemical probes of DNA conformation. The results have indicated that the attachment of distamycin to cisplatin mainly affects the sites involved in the interstrand cross-links so that these adducts are preferentially formed between complementary guanine and cytosine residues. This interstrand cross-link bends the helix axis by approximately 35 degrees toward minor groove, unwinds DNA by approximately 95 degrees and distorts DNA symmetrically around the adduct. In addition, CD spectra of restriction fragments modified by the cisplatin-distamycin conjugates have demonstrated that distamycin moiety in the interstrand cross-links of these compounds interacts with DNA. This interaction facilitates the formation of these adducts. Hence, the structural impact of the specific interstrand cross-link detected in this study deserves attention when biological behavior of cisplatin derivatives targeted by oligopeptide DNA minor-groove-binders is evaluated.     

DNA interstrand cross-links of the novel antitumor trinuclear platinum complex BBR3464. Conformation,recognition by high mobility group domain proteins,and nucleotide excision repair

The novel phase II antitumor polynuclear platinum drug BBR3464 ([(trans-PtCl(NH(3))(2))(2)(mu-trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2))](NO(3))(4)) forms intra- and interstrand cross-links (CLs) on DNA (which is the pharmacological target of platinum drugs). We examined first in our recent work how various intrastrand CLs of BBR3464 affect the conformation of DNA and its recognition by cellular components (Zehnulova, J., Kasparkova, J., Farrell, N., and Brabec, V. (2001) J. Biol. Chem. 276, 22191-22199). In the present work, we have extended the studies on the DNA interstrand CLs of this drug. The results have revealed that the interstrand CLs are preferentially formed between guanine residues separated by 2 base pairs in both the 3' --> 3' and 5' --> 5' directions. The major 1,4-interstrand CLs distort DNA, inducing a directional bending of the helix axis and local unwinding of the duplex. Although such distortions represent a potential structural motif for recognition by high mobility group proteins, these proteins do not recognize 1,4-interstrand CLs of BBR3464. On the other hand, in contrast to intrastrand adducts of BBR3464, 1,4-interstrand CLs are not removed from DNA by nucleotide excision repair. It has been suggested that interstrand CLs of BBR3464 could persist considerably longer in cells compared with intrastrand adducts, which would potentiate the toxicity of the interstrand lesions to tumors sensitive to this polynuclear drug.     

Covalent binding and interstrand cross-linking of duplex DNA by dirhodium(II,II) carboxylate compounds

The study of the interactions of double-stranded (ds) DNA with the dirhodium carboxylate compounds Rh(2)(O(2)CCH(3))(4)(H(2)O)(2) (Rh1), [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2) (Rh2), and Rh(2)(O(2)CCF(3))(4) (Rh3) supports the presence of covalently linked DNA adducts, including stable DNA interstrand cross-links. The present biochemical study refutes earlier claims that no reaction between dirhodium compounds and dsDNA occurs. The reversal behavior of these interstrand cross-links in 5 M urea at 95 degrees C (for different heating times) implies the presence of various coordination modes involving ax/ax, ax/eq, and eq/eq DNA interactions with the dirhodium core. The reaction rates of the dirhodium compounds with dsDNA were determined spectroscopically and are in the order Rh1 < Rh2 < Rh3. This difference in behavior of the three dirhodium compounds correlates with the lability of the leaving groups and corresponds to the extent of interstrand cross-link formation by these compounds on a 123 bp DNA fragment, as observed by denaturing polyacrylamide gel electrophoresis (dPAGE). Since all three dirhodium compounds form covalent Rh-DNA adducts, including interstrand cross-links, it is important that DNA be considered a potential target for biological activity of these dirhodium carboxylate compounds.     

Contribution of XPF functional domains to the 5' and 3' incisions produced at the site of a psoralen interstrand cross-link.

XPF forms a heterodimeric complex with ERCC1 and is required for the repair of DNA interstrand cross-links. In association with ERCC1, it is involved in production of the 5' incision at the site of a psoralen interstrand cross-link as well as the 3' incision. The present study was carried out to determine the functional domains of XPF that are important in the production of the 5' and 3' incisions that occur at a site of a psoralen interstrand cross-link. Monoclonal antibodies (mAbs) were utilized that had been generated against polypeptide fragments of XPF and affinity-mapped to specific regions of XPF. These mAbs were examined for their ability to differentially inhibit production of dual incisions in DNA by normal human chromatin-associated protein extracts that contain XPF and ERCC1. These studies show that two regions of XPF, one N-terminal region from amino acids 12-166 and one C-terminal region from amino acids 702-854, are the most important in the production of the 5' incision. The same N-terminal region and the C-terminal region from amino acids 702-916 are also involved in the 3' incision, though to a much lesser extent. Since this C-terminal region corresponds to the proposed site of interaction of ERCC1 with XPF, these results suggest that binding of ERCC1 to XPF is critical for its ability to produce the 5' and 3' incisions at the site of an interstrand cross-link, possibly through activation or regulation of the endonucleolytic activity of the N-terminal domain of XPF.     

Structure of the 1,4-bis(2'-deoxyadenosin-N6-yl)-2R,3R-butanediol cross-link arising from alkylation of the human N-ras codon 61 by butadiene diepoxide

The solution structure of the 1,4-bis(2'-deoxyadenosin-N(6)-yl)-2R,3R-butanediol cross-link arising from N(6)-dA alkylation of nearest-neighbor adenines by butadiene diepoxide (BDO(2)) was determined in the oligodeoxynucleotide 5'-d(CGGACXYGAAG)-3'.5'-d(CTTCTTGTCCG)-3'. This oligodeoxynucleotide contained codon 61 (underlined) of the human N-ras protooncogene. The cross-link was accommodated in the major groove of duplex DNA. At the 5'-side of the cross-link there was a break in Watson-Crick base pairing at base pair X(6).T(17), whereas at the 3'-side of the cross-link at base pair Y(7).T(16), base pairing was intact. Molecular dynamics calculations carried out using a simulated annealing protocol, and restrained by a combination of 338 interproton distance restraints obtained from (1)H NOESY data and 151 torsion angle restraints obtained from (1)H and (31)P COSY data, yielded ensembles of structures with good convergence. Helicoidal analysis indicated an increase in base pair opening at base pair X(6).T(17), accompanied by a shift in the phosphodiester backbone torsion angle beta P5'-O5'-C5'-C4' at nucleotide X(6). The rMD calculations predicted that the DNA helix was not significantly bent by the presence of the four-carbon cross-link. This was corroborated by gel mobility assays of multimers containing nonhydroxylated four-carbon N(6),N(6)-dA cross-links, which did not predict DNA bending. The rMD calculations suggested the presence of hydrogen bonding between the hydroxyl group located on the beta-carbon of the four-carbon cross-link and T(17) O(4), which perhaps stabilized the base pair opening at X(6).T(17) and protected the T(17) imino proton from solvent exchange. The opening of base pair X(6).T(17) altered base stacking patterns at the cross-link site and induced slight unwinding of the DNA duplex. The structural data are interpreted in terms of biochemical data suggesting that this cross-link is bypassed by a variety of DNA polymerases, yet is significantly mutagenic [Kanuri, M., Nechev, L. V., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580].     

Interstrand cross-links and sequence specificity in the reaction of cis-dichloro(ethylenediamine)platinum(II) with DNA

Characterization of the adducts produced in DNA by the cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) and a radiolabeled analogue, [3H]-cis-dichloro(ethylenediamine)platinum(II) ([3H]-cis-DEP) was recently reported [Eastman, A. (1983) Biochemistry 22, 3927]. Both drugs reacted at identical sites in DNA, most of which produced intrastrand cross-links. DNA-interstrand cross-links, which represent less than 1% of total platination, have now been characterized. DNA containing interstrand cross-links was enriched for on the basis of its renaturability after boiling. This DNA was digested to deoxyribonucleosides, and the adducts were separated by high-pressure liquid chromatography. A cross-link between two deoxyguanosines was observed to be the most prominent adduct. It is proposed that the major sequence in which this cross-link occurs is 5'-CG-3'. DNA that was incubated with [3H]-cis-DEP for 1 h showed low levels of interstrand cross-links. After removal of unreacted drug, their frequency increased significantly over 6 h with a maximum occurring at about 12 h. A similar phenomenon was seen in the case of intrastrand cross-links that contained adenine, in particular when the cross-link was between the terminal bases in an ANG trinucleotide sequence (N is any nucleotide). The primary site of reaction is at guanine, with a slow subsequent cross-link to the adenine. A model is presented that is consistent with the observation that adenine is always at the 5' terminus of these adducts. The proportion of adducts at ANG sequences also increased at elevated temperatures. This is discussed with regard to potential significance during hyperthermia treatment of patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of the R(61,2)- and S(61,2)-alpha-(N6-adenyl)styrene oxide adducts on the A.C mismatched base pair in an oligodeoxynucleotide containing the human N-ras codon 61.

Conformational studies of R- and S-alpha-(N6-adenyl)styrene oxide adducts mismatched with deoxycytosine at position X6 in d(CGGACXAGAAG).d(CTTCTCGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, are described. These were the R- and S(61,2)C adducts. The S(61,2)C adduct afforded a stable solution structure, while the R(61,2)C adduct resulted in a disordered structure. Distance restraints for the S(61, 2)C adduct were calculated from NOE data using relaxation matrix analysis. These were incorporated as effective potentials into the total energy equation. The structures were refined using restrained molecular dynamics calculations which incorporated a simulated annealing protocol. The accuracy of the emergent structures was evaluated by complete relaxation matrix methods. The structures refined to an average rms difference of 1.07 A, determined by pairwise analysis. The experimentally determined structure was compared to NOE intensity data using complete relaxation matrix back-calculations, yielding an R1x value of 11.2 x 10(-)2. The phenyl ring of the styrene in the S(61,2)C adduct was in the major groove and remained oriented in the 3'-direction as observed for the corresponding S(61,2) adduct paired with thymine [Feng, B., Zhou, L., Pasarelli, M., Harris, C. M., Harris, T. M., and Stone, M. P. (1995) Biochemistry 34, 14021-14036]. A shift of the modified adenine toward the minor groove resulted in the styrenyl ring stacking with nucleotide C5 on the 5'-side of the lesion, which shifted toward the major groove. Unlike the unmodified A.C mismatch, neither the S(61,2)C nor the R(61,2)C adduct formed protonated wobble A.C hydrogen bonds. This suggests that protonated wobble A.C pairing need not be prerequisite to low levels of alpha-SO-induced A --> G mutations. The shift of the modified adenine toward the minor groove in the S(61,2)C structure may play a more important role in the genesis of A --> G mutations. The disordered structure of the R(61,2)C adduct provides a potential explanation as to why that adduct does not induce A --> G mutations.     

UvrABC incision of N-methylmitomycin A-DNA monoadducts and cross-links

The Escherichia coli UvrABC endonuclease is a multisubunit enzyme that initiates the repair of a wide variety of DNA lesions in vivo by making dual incisions on a damaged strand at the eighth or ninth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the modified base. It has been hypothesized that UvrABC is able to recognize a broad spectrum of lesions because it does not recognize the lesion per se but rather gross helical distortions that the lesion induces in the DNA. Several lesions have recently been studied which are thermal stabilizing and are not believed to distort the DNA grossly, including the CC-1065-N-3-adenine and anthramycin-N-2-guanine adducts. We have studied the activity of UvrABC in vitro on another thermal stabilizing and nondistortive adduct, N-methylmitomycin A (NMA), a bifunctional DNA-alkylating agent that reacts with guanine on the side facing the minor groove, yielding either monoadducts or interstrand cross-links. NMA adducts increase the thermal stability of DNA, and theoretical calculations indicate that NMA adducts do not grossly distort the DNA helix. Our results show that UvrABC makes incisions at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to an NMA monoadduct, consistent with the incision pattern observed for the majority of other lesions that are also recognized by UvrABC. DNA containing a site-specific NMA cross-link was also recognized and incised by UvrABC. The rate of incision of NMA cross-linked DNA was about 200-fold higher in supercoiled molecules than in relaxed molecules, whereas the rate of incision of DNA containing NMA monoadducts was stimulated approximately 2-fold by supercoiling. The signal for UvrABC recognition and incision of damaged DNA is discussed in relation to the ability of UvrABC to incise NMA adducts as well as other nondistortive lesions.     

Repair of an interstrand DNA cross-link initiated by ERCC1-XPF repair/recombination nuclease

Interstrand DNA cross-link damage is a severe challenge to genomic integrity. Nucleotide excision repair plays some role in the repair of DNA cross-links caused by psoralens and other agents. However, in mammalian cells there is evidence that the ERCC1-XPF nuclease has a specialized additional function during interstrand DNA cross-link repair, beyond its role in nucleotide excision repair. We placed a psoralen monoadduct or interstrand cross-link in a duplex, 4-6 bases from a junction with unpaired DNA. ERCC1-XPF endonucleolytically cleaved within the duplex on either side of the adduct, on the strand having an unpaired 3' tail. Cross-links that were cleaved only on the 5' side were purified and reincubated with ERCC1-XPF. A second cleavage was then observed on the 3' side. Relevant partially unwound structures near a cross-link may be expected to arise frequently, for example at stalled DNA replication forks. The results show that the single enzyme ERCC1-XPF can release one arm of a cross-link and suggest a novel mechanism for interstrand cross-link repair.     

Defects in interstrand cross-link uncoupling do not account for the extreme sensitivity of ERCC1 and XPF cells to cisplatin

The anticancer drug cisplatin reacts with DNA leading to the formation of interstrand and intrastrand cross-links that are the critical cytotoxic lesions. In contrast to cells bearing mutations in other components of the nucleotide excision repair apparatus (XPB, XPD, XPG and CSB), cells defective for the ERCC1-XPF structure-specific nuclease are highly sensitive to cisplatin. To determine if the extreme sensitivity of XPF and ERCC1 cells to cisplatin results from specific defects in the repair of either intrastrand or interstrand cross-links we measured the elimination of both lesions in a range of nucleotide excision repair Chinese hamster mutant cell lines, including XPF- and ERCC1-defective cells. Compared to the parental, repair-proficient cell line all the mutants tested were defective in the elimination of both classes of adduct despite their very different levels of increased sensitivity. Consequently, there is no clear relationship between initial incisions at interstrand cross-links or removal of intrastrand adducts and cellular sensitivity. These results demonstrate that the high cisplatin sensitivity of ERCC1 and XPF cells likely results from a defect other than in excision repair. In contrast to other conventional DNA cross-linking agents, we found that the repair of cisplatin adducts does not involve the formation of DNA double-strand breaks. Surprisingly, XRCC2 and XRCC3 cells are defective in the uncoupling step of cisplatin interstrand cross-link repair, suggesting that homologous recombination might be initiated prior to excision of this type of cross-link.     

Intrastrand DNA cross-links as tools for studying DNA replication and repair: two-, three-, and four-carbon tethers between the N(2) positions of adjacent guanines

A general protocol for preparation of oligonucleotides containing intrastrand cross-links between the exocyclic amino groups of adjacent deoxyguanosines has been developed. A series of 2, 3, and 4 methylene cross-links was incorporated site-specifically into an 11-mer (5'-GGCAGGTGGTG-3', cross-linked positions are underlined) via a reaction between oligonucleotide containing 2-fluoro-O(6)-trimethylsilylethyl deoxyinosines and the appropriate diamine (ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane). These cross-linked-oligonucleotides were studied for their ability to bend DNA by the method of Koo and Crothers [Koo, H. S., and Crothers, D. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1763-1767] in which the mobility of ligated oligomers in nondenaturing polyacrylamide gels is evaluated. It was found that all cross-links induced bending (2-carbon cross-link, 30.0 +/- 4.0 deg/turn; 3-carbon cross-link, 11.7 +/- 1.6 deg/turn; 4-carbon cross-link, 7.4 +/- 1.0 deg/turn). Despite the differing extent of helical distortion exhibited by the cross-links, all appeared to be equally blocking to replication by the Escherichia coli polymerases, pol I, pol II, and pol III. In contrast, when incision of the cross-links by the E. coli UvrABC nucleotide incision complex was studied, the extent of incision of the cross-link was found to correlate closely with the degree of bending measured in the gel mobility assay, i.e., the efficiency of incision was 2-carbon > 3-carbon > 4-carbon.     

Interstrand cross-linking of DNA by FK317 and its deacetylated metabolites FR70496 and FR157471

FR900482 (1) and FR66979 (2) are structurally novel natural products isolated by Fujisawa in 1987 and have been shown to be highly potent antitumor antibiotics structurally related to the mitomycins. Studies on the mode of action have established that these new agents form covalent DNA interstrand cross-links both in vitro and in vivo as a result of the reactive mitosene intermediate generated upon bioreductive activation. Semisynthetic analogues such as FK973 (3) and FK317 (4) were developed in the search for potentially superior clinical candidates. Although FK317 has been shown to be a potent compound, to date no direct evidence of DNA interstrand cross-link sequence specificity has been reported. In this study, DNA interstrand cross-links were generated by treatment of a synthetic duplex DNA substrate with FK317 (4) and its deacetylated metabolites FR70496 (5) and FR157471 (6). Analysis by gel electrophoresis revealed the formation of orientation isomers displaying electrophoretic mobility vastly greater than the mobilities of those generated from FR900482 (1). Despite these differences, it was established by Fe(II)-EDTA footprinting that FK317 (4) as well as 5 and 6 forms DNA interstrand cross-links within the expected 5'CpG3' step, clearly demonstrating that the phenolic hydrogen in 1 and 2 is not a prerequisite for efficient DNA interstrand cross-linking by the FR class of compounds.     

Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions 5' to the cross-linked base and removes a 22- to 28-nucleotide-long damage-free strand.

Most DNA repair mechanisms rely on the redundant information inherent to the duplex to remove damaged nucleotides and replace them with normal ones, using the complementary strand as a template. Interstrand cross-links pose a unique challenge to the DNA repair machinery because both strands are damaged. To study the repair of interstrand cross-links by mammalian cells, we tested the activities of cell extracts of wild-type or excision repair-defective rodent cell lines and of purified human excision nuclease on a duplex with a site-specific cross-link. We found that in contrast to monoadducts, which are removed by dual incisions bracketing the lesion, the cross-link causes dual incisions, both 5' to the cross-link in one of the two strands. The net result is the generation of a 22- to 28-nucleotide-long gap immediately 5' to the cross-link. This gap may act as a recombinogenic signal to initiate cross-link removal.     

The exocyclic 1,N2-deoxyguanosine pyrimidopurinone M1G is a chemically stable DNA adduct when placed opposite a two-base deletion in the (CpG)3 frameshift hotspot of the Salmonella typhimurium hisD3052 gene.

The pyrimidopurinone adduct M1G [3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-a]-purin-10(3H)-one], formed in DNA upon exposure to malondialdehyde or base propenals, was incorporated into 5'-d(ATCGCMCGGCATG)-3'-5'-d(CATGCCGCGAT)-3', where M = M1G. This duplex contained a two-nucleotide bulge in the modified strand, and was named the M1G-2BD oligodeoxynucleotide. It provided a model for -2 bp strand slippage deletions associated with the (CpG)3-iterated repeat hotspot for frameshift mutations from the Salmonella typhimurium hisD3052 gene. M1G was chemically stable in the M1G-2BD duplex at neutral pH. The two-base bulge in the M1G-2BD oligodeoxynucleotide was localized and consisted of M1G and the 3'-neighbor deoxycytosine. The intrahelical orientation of M1G was established from a combination of NOE and chemical shift data. M1G was in the anti conformation about the glycosyl bond. The 3'-neighbor deoxycytosine appeared to be extruded toward the major groove. In contrast, when M1G was placed into the corresponding fully complementary (CpG)3-iterated repeat duplex at neutral pH, spontaneous and quantitative ring-opening to N(2)-(3-oxo-1-propenyl)-dG (the OPG adduct) was facilitated [Mao, H., Reddy, G. R., Marnett, L. J., and Stone, M. P. (1999) Biochemistry 38, 13491-13501]. The structure of the M1G-2BD duplex suggested that the bulged sequence lacked a cytosine amino group properly positioned to facilitate opening of M1G and supports the notion that proper positioning of deoxycytosine complementary to M1G is necessary to promote ring-opening of the exocyclic adduct in duplex DNA. The structure of the M1G-2BD duplex was similar to that of the structural analogue 1,N(2)-propanodeoxyguanosine (PdG) in the corresponding PdG-2BD duplex [Weisenseel, J. P., Moe, J. G., Reddy, G. R., Marnett, L. J., and Stone, M. P. (1995) Biochemistry 34, 50-64]. The fixed position of the bulged bases in both instances suggests that these exocyclic adducts do not facilitate transient bulge migration.     

In vitro repair of psoralen-DNA cross-links by RecA, UvrABC, and the 5'-exonuclease of DNA polymerase I

Psoralens produce DNA interstrand cross-links which are thought to be repaired via a sequential excision and recombination mechanism in Escherichia coli. The first round of incision by UvrABC has been characterized: it results in 11-base oligonucleotide cross-linked to an intact DNA strand (Van Houten, B., Gamper, B., Holbrook, S.R., Hearst, J.E., and Sancar, A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8077-8081). In the present work, DNA substrates containing 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) cross-links in defined positions are constructed and used to analyze the other steps in repair. It is shown that RecA protein mediates strand transfer past an oligonucleotide cross-linked to a single-stranded DNA circle and that the resulting heteroduplex is a substrate for the UvrABC complex: it excises a double-stranded oligonucleotide which contains the HMT cross-link. It is also found that the first round of UvrABC incision does not lead directly to strand exchange but that an intervening step is needed. That step is carried out in vitro by the 5'-exonuclease activity of DNA polymerase I (pol I) which creates a single-stranded DNA region (a gap) at an incised cross-link such that RecA can initiate strand exchange. Studies using cross-linked oligonucleotides showed that the gap produced by pol I results from the inability of the polymerase to add nucleotides to a 3'-OH end two to three nucleotides away from the furan side of an HMT cross-link. Pol I can, however, extend a 3'-OH end next to the pyrone side of the cross-link. Since UvrABC incises predominantly the furan side of psoralen cross-links in duplex DNA, this discrepancy has important consequences for repair.